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Siddardha et al. World Journal of Pharmaceutical Research
ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR
THE SIMULTANEOUS ESTIMATION OF IRBESARTAN AND
HYDROCHLOROTHIAZIDE IN PHARMACEUTICAL DOSAGE
FORM BY RP-HPLC
M.Siddardha*, K.Mangamma And G.Manikumar
Department of Pharmaceutical Analysis, University College of Pharmacy, JNTU, Kakinada,
E.G.Dist, A.P, India.
ABSTRACT
A simple, specific, accurate and precise reverse phase high pressure
liquid chromatographic method has been developed for the
simultaneous determination of Irbesartan and Hydrochlorothiazide in
tablet dosage form by using Hypersil BDS C18, 250mm X 4.6 mm, 5µm
column. The samples were analyzed by using Methanol: water (80: 20)
v/v as a mobile phase at the flow rate of 1.0 ml/min in isocratic mode
and detection wavelength is 257 nm. Both the drugs were eluted within
5 minutes and gave sharp peaks with high theoretical plate count and
low tailing factor. The retention time for Irbesartan and
Hydrochlorothiazide was found to be 2.872 and 3.943 min
respectively. The validation was carried according to ICH guidelines.
Calibration curve was linear with correlation coefficient of 0.997 and
0.999 over a concentration range of 60µg/mL-240µg/mL and 5-20µg/ml for Irbesartan and
Hydrochlorothiazide respectively. The percent recovery was 99.80 for Irbesartan and 99.80
for Hydrochlorothiazide indicating accuracy and reliability of method. So the method can be
used for estimation of combination of these drugs in tablet dosage form.
Key words: Irbesartan, Hydrochlorothiazide, RP-HPLC
1. INTRODUCTION
Irbesartan is a non-peptide compound, chemically described as a 2-butyl-3-[p-(o-1H-tetrazol-
5ylphenyl) benzyl]-1,3-diazaspiro[4.4]non-1-en-4-one. Its empirical formula is C25H28N6O,
World Journal of Pharmaceutical ReseaRch
Volume 3, Issue 1, 1060-1075. Research Article ISSN 2277 – 7105
Article Received on 21 October2013 Revised on 23 November 2013, Accepted on 26 December 2013
*Correspondence for
Author:
M.Siddardha,
Department of Pharmaceutical
Analysis, University College of
Pharmacy, JNTU, Kakinada,
E.G.Dist, A.P, India.
venky224@gmail.com
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and its structural formula is given in Fig.1. Hydrochlorothiazide is 6-chloro-3, 4-dihydro-2H-
1, 2, 4-benzothiadiazine-7-sulfonamide 1,1dioxide. Its empirical formula is
C7H8ClN3O4S2 and its structural formula is given in Fig.2. Irbesartan is a type of medicine
called an angiotensin II receptor antagonist. It works by preventing the action of a hormone in
the body called angiotensin II. Irbesartan blocks the receptors that angiotensin II acts on, and
so prevents its actions. The main result of this is that the peripheral blood vessels are allowed
to widen, which means that there is more space and less resistance in these blood vessels.
This lowers the pressure inside the blood vessels.
Hydrochlorothiazide is a type of medicine known as a thiazide diuretic. Thiazide diuretics act
in the kidneys, where they increase the production of urine. They work by causing the
kidneys to increase the amount of salts, such as potassium and sodium that are filtered out of
the blood and into the urine. When these salts are filtered out of the blood by the kidneys,
water is also drawn alongside. As hydrochlorothiazide increases the removal of salts from the
blood, it also causes more water to be drawn out of the blood and into the urine.
A combination of 3000mg of Irbesartan and 12.5mg Hydrochlorothiazide is available
commercially as tablets. This combination is used in the treatment of Hypertension.
Literature survey revealed that UV spectrophotometric, HPLC methods, HPTLC and
Capillary electrophoresis methods are available for estimation of MET and FENO
individually and also in combination with other drugs [6-15]. Few reports are also available
on the simultaneous estimation of Irbesartan and Hydrochlorothiazide using techniques like
Spectrophotometric and RP-HPLC but they are suffering from their own drawbacks like high
retention time or poor resolution or use of expensive solvents etc.
The aim of present work is to develop and validate a new, simple, better and economical
method for the simultaneous estimation of Irbesartan and Hydrochlorothiazide in tablet
dosage form by RP-HPLC with improved conditions and parameters for routine use in the
laboratories. The chemical structures of the assayed compounds are given below.
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Fig-2 Structure of Irbesartan
Fig-3 Structure of Hydrochlorothiazide
2. Reagents and materials
Pure drugs samples of Irbesartan and Hydrochlorothiazide were obtained as a gift samples
from Aurobindo Pharma Ltd, Hyderabad, Andhrapradesh, India. HPLC grade methanol,
acetonitrile were procured from Merck. In-house Millipore water was used throughout the
study.
3. Instrumentation
The apparatus used in this study were Waters Alliance E2690 with empower 2 software and
UV-2489 detector with auto sampler HPLC.UV Double Beam spectrometer systronics UV
3000 was used to find the lambda maximum of the drugs. ADWA AD102 U pH meter,
ENERTECH SE60US ultrasonicator were also used.
Table-1 Chromatographic conditions
Parameters Optimised condition
Mobile Phase Methanol:water(80:20)
Column type Hypersil BDS C18, 250mm X 4.6 mm, 5µ
Flow rate 1.0ml/min
Detection wavelength UV at 257nm
Temperature Ambient
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Injection volume 20µl
Retention time Irbesartan-2.86min,Hydrochlorothiazide-3.94min
4. Determination of working wavelength (λ max)
Fig-3 Overlay spectra of both Irbesartan and Hydrochlorothiazide
To ascertain the maximum wavelength (λ max), the standard solutions were scanned between
200-400nm in UV spectrometer and the wavelength of 257nm is best suitable for both the
analytes (Irbesartan and Hydrochlorothiazide) and hence the experiment is carried out in the
wavelength of 257nm.
5. Preparation of solutions
5.1. Preparation of Mobile phase
800mL (80%) of methanol is mixed with the 200mL (20%) of nanopure water and filtered
through vacuum filtration using 0.45µm filter and degassed in a ultrasonic water bath for
10min.
5.2. Preparation of Diluent
Mobilephase was used as diluent.
5.3. Preparation of Standard stock solution
Standard stock solutions of Irbesartan and hydrochlorothiazide were prepared by dissolving
300mg of Irbesartan and25mg of hydrochlorothiazide were dissolved in 20 mL of diluent
separately and sonicated for 10min and made up the volume to 100mL with diluent and were
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labelled as standard stock solution-1(Irbesartan 3mg/mL) and standard stock solution-
2(hydrochlorothiazide 0.25mg/mL).
5.4. Preparation of mixed standard solution
0.4mL of standard stock solution-1, 0.4mL of standard stock solution-2 was transferred to 10
mL volumetric flask and made up the volume to 10mL with diluent.
5.5. Preparation of sample solution
Twenty tablets were weighed, ground in to a fine powder and mixed thoroughly. A quantity
of powder equivalent to 300mg of Irbesartan (25mg of hydrochlorothiazide) was weighed and
transferred in to a 100ml volumetric flask and was dissolved in the diluents. The volume was
made up to the mark with the same and the resulting solution was labelled as test stock
solution. A solution containing 120µg/ml of Irbesartan (10µg/ml of hydrochlorothiazide) was
prepared by appropriate dilution of the test stock solution. The solutions thus prepared were
filtered through 0.45µ membrane filter and the filtrate was sonicated for 10min.
6. Assay
20µL of test solution containing (120µg/mL of Irbesartan and 10µg/mL of
Hydrochlorothiazide) was injected three times at the optimised method conditions and the
chromatograms of three injections were recorded and the % Assay was calculated using the
following formula:
%Assay= Sample Area x Standard Wt x Tablet Avg Wt x 100
Standard Area x Sample Wt x Label Amt
7. Method validation
The developed method is validated by following the ICH guidelines with respect to
parameters such as system suitability, linearity, accuracy, precision, limit of detection, limit
of quantification, robustness and specificity.
7.1. System Suitability
To verify the working condition of analytical system whether it can give accurate and precise
results, 20µL of working standard solution containing 120µg/mL of Irbesartan and 10µg/mL
of hydrochlorothiazide was injected for 5 times and the chromatograms were taken for the
same and the results were tabulated.
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7.2. Linearity
The linearity of the Irbesartan and Hydrochlorothiazide was established over the range of
60µg/mL-240µg/mL and 5-20µg/mL respectively and the chromatograms were taken for the
same and calibration plot was drawn and regression analysis was performed and regression
coefficient was calculated.
7.3. Accuracy
The accuracy of the method was evaluated by recovery studies which were carried out by
standard addition method, where a known amount of sample was spiked with mixed standard
solution at three levels 50%,100% and 150%.At each levels recovery studies were performed
in triplicate and expressed as percentage recoveries.
7.3.1. Preparation of mixed standard solution
30mg of Irbesartan and 2.5mg oh hydrochlorothiazide were accurately weighed in to a 10 mL
volumetric flask and made up the volume with diluent. The resulting solution contains the
3000µg/mL of Irbesartan and 250µg/mL of hydrochlorothiazide.
7.3.2. Preparation of standard and test solutions
Mixed standard solutions containing 40, 80 and 120µg/mL of Irbesartan (5, 10 and 15µg/mL
of hydrochlorothiazide) respectively were prepared in triplicates, from the mixed standard
stock solution by appropriate dilutions. Sample solutions containing 40µg/mL of Irbesartan
(5µg/mL of hydrochlorothiazide) were prepared by appropriate dilution of sample stock
solution (containing 3000 µg/mL of Irbesartan and 250 µg/mL of hydrochlorothiazide).
7.3.3. Procedure for spiking
Spiking at 50%level was accomplished in triplicate, by adding 3ml of unfilterated sample
solution to 1.5mL of mixed standard stock solution in a test tube. The contents of the test tube
were shaken for some time and filtered through a whatmann filter in to a 10mL volumetric
flask and washed 2 times with diluent and made up the volume with the diluent and filtered
through 0.44 µm filter in to autoinjector vial.In same way the 100% and 150% spiking was
prepared by adding 3mL of unfiltered sample solution in to 3mL and 4.5mL of mixed
standard stock solution respectively in to a separate test tubes.
%Recovery = ((b-a)/c)*100
Where a=response of test solution
b=response of spiked solution
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c=response of standard solution
7.4. Precision
The precision was checked at levels of repeatability and also checked for both system
precision and method precision. In case of system precision only one preparation was done at
the level of 120µg/mL, injected on the same day for 5times and where as for interday
precision it was determined by injecting by preparing on different days. In case of method
precison five different preparations were done and both intra and inter day precison was
performed. The results of the repeatability and intermediate precision were calculated and
tabulated.
7.5. Limit of detection
7.2µg/mL of Irbesartan and 2µg/mL of hydrochlorothiazide was prepared by making
appropriate dilutions from standard stock solutions and injected in to HPLC system and
optimised chromatographic conditions; using signal to noise ratio, LOD was calculated.
LOD=3.3(σ/S)
σ = standard deviation
S=Slope of the calibration curve
7.6. Limit of quantification
18µg/mL of Irbesartan and 3.2µg/mL of hydrochlorothiazide was prepared by making
appropriate dilutions from standard stock solutions and injected in to HPLC system and
optimised chromatographic conditions, signal using signal to noise ratio, LOQ was calculated
LOD=10(σ/S)
σ = standard deviation
S=Slope of the calibration curve
7.7. Robustness
Robustness was performed by making deliberate changes in the chromatographic conditions
like
1) Change in flow rate (±0.2mL/min)
2) Change in detection wavelength (±2nm)
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7.8. Specificity
Specificity was performed by injecting placebo which was used for preparation of test
solution. This placebo was filtered through 0.44µm filter and injected in to HPLC system.
8. RESULTS AND DISCUSSION
To develop a precise, accurate and suitable RP-HPLC method for the estimation of Irbesartan
and Hydrochlorothiazide, different mobile phases were tried but the proposed
chromatographic conditions were found to be appropriate for quantitative determination.
Irbes
arta
n - 2
.869
Hyd
roch
loro
thia
zide
- 3.
942
AU
0.00
0.05
0.10
0.15
0.20
0.25
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Fig-4 Chromatogram of standard
Irbes
arta
n - 2
.869
Hyd
roch
loro
thia
zide
- 3.
942
AU
0.00
0.05
0.10
0.15
0.20
0.25
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Fig-5 Chromatogram of sample
AU
-0.008
-0.006
-0.004
-0.002
0.000
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Fig-6 Chromatogram of placebo
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8.1. Assay
The assay was performed on the tablet formulation and the % drug contents of Irbesartan and
hydrochlorothiazide were found to be 99.80 and 99.80% which were in acceptance limits
Table-2 Results of Assay
Parameters Irbesartan Hydrochlorothiazide
Sample Area 1246839 546612
Standard Area 1246748 546597
Standard Wt 300 25
Sample Wt 501 501
Average tablet wt 500 500
Label claim 300 25
%Assay 99.80 99.80
8.2. System suitability
The system suitability of the present method was accomplished from the resolution,
theoretical plates, assymetry factor of Irbesartan and hydrochlorothiazide at the optimised
conditions. The parameters were recorded and tabulated and were found to be within the
acceptance criteria.
Table-3 System suitability data of Irbesartan and hydrochlorothiazide
S.No Name Retention Time
Area Height USP Tailing
USP Plate count
1 Irbesartan 2.872 1246426 252167 1.12 8086.89
2 Hydrochlorothiazide 3.943 546725 91227 1.08 9873.71
8.3. Linearity
The linearity of the Irbesartan and Hydrochlorothiazide was established over the range of
60µg/mL-240µg/mL and 5-20µg/mL respectively and correlation coefficient of the respective
calibration curves were 0.997and 0.999 for Irbesartan and hydrochlorothiazide respectively
which were well within the acceptance criteria.
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Table-4 Linearity results for Irbesartan and Hydrochlorothiazide
Standard Irbesartan Hydrochlorothiazide
Concentration (µg/mL) Peak area Concentration
(µg/mL) Peak area
CC1 60 623030 5 273334
CC2 90 935298 7.5 410569
CC3 120 1246810 10 546585
CC4 150 1557240 12.5 683562
CC5 180 1870197 15 820392
CC6 210 2182190 17.5 947307
CC7 240 2394180 20 1094198
Statistica
l analysis
Slope -- 10036 -- 54427
Y-intercept -- 38667 -- 1943
Correlation
coefficient(r2) -- 0.997 -- 0.999
Fig-6Linearity curve of Irbesartan
Fig-7 Linearity curve of Hydrochlorothiazide
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8.4. Accuracy
The accuracy of the present method was evaluated from recovery studies, by standard
addition method which is performed at three levels of 50,100&150% levels. The mean
percentage recoveries at each level of Irbesartan and Hydrochlorothiazide were found to be
98.60 to 101.30 and 99.13 to 101.73 respectively.
Table-5 Accuracy data for Irbesartan
Spiked levels
Standard sample Spiked %Recovery
Mean % recovery Conc
(µg/mL) Peak area
Conc (40µg/mL) Peak area
Conc (µg/mL) Peak area
50 40
440111 430003
80
867895 97.98 98.66
441209 440034 872376 98.75
440093 440003 873540 99.27
100 80
841506
Mean
120
1285768 100.90 101.30
841517 1275768 99.71
841530 1305768 103.27
150 120
1251221
160
1690890 100.24
99.33 1262683 1687890 99.09
1271224 436680 1690890 98.66
Table-6 Accuracy data for Hydrochlorothiazide
Spiked levels
Standard Sample Spiked %Rec
overy
Mean %
recovery Conc (µg/mL)
Peak area
Conc (5µg/mL) Peak area
Conc (µg/mL) Peak area
50 5
273246 273557
10
547547 99.38
99.13 275465 275437 547980 98.74
276547 278965 550480 99.26
100 10
546474
Mean
15
836268 102.53
101.73 544568 822376 100.33
554365 843246 102.33
150 15
836578
20
1114915 100.28
99.68 862376 1134150 99.51
843246 275986 1112893 99.25
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8.5. Precision
The precision of the proposed method was established from the %RSDs of the percentage
assays of the drugs at the levels of repeatability (intra-day) and intermediate precision
(interday) and also at system and method precision. In system precision, the %RSDs of
Irbesartan and hydrochlorothiazide at intra-day precision were found to be 0.035% and
0.029% respectively, at inter-day precision were found to be 0.015 and 0.022% respectively.
In method precision, the %RSDs of Irbesartan and hydrochlorothiazide at intra-day precision
were found to be 0.067% and 0.041% respectively, at inter-day precision were found to be
0.012 and 0.036% respectively. As the %RSDs were found to be within the acceptance limit
(%RSD < 2%) at all the levels, the proposed method was said to be precise.
Table-7 Intra day and inter day precision data for System precision
Injection
No.
Intraday(peak area) Inter day(peak area)
Irbesartan Hydrochlorothiazide Irbesartan Hydrochlorothiazide
Injection-1 1246538 546435 1246650 546591
Injection-2 1246634 546876 1246974 546647
Injection-3 1246738 546647 1246556 546342
Injection-4 1246876 546576 1246889 546497
Injection-5 1247645 546675 1246997 546435
Average 1246886 546641.8 1246813 546650
Standard
deviation 442.349 160.5263 198.8535 121.510
%RSD 0.035 0.029 0.015 0.022
Table-8 Intra day and inter day precision data for Method Precision
Preparation
No.
Intraday(peak area) Inter day(peak area)
Irbesartan Hydrochlorothiazide Irbesartan Hydrochlorothiazide
Prep-1 1246982 546672 1246812 546342
Prep-2 1246567 546545 1246538 546687
Prep-3 1245345 546342 1246876 546456
Prep-4 1247657 546223 1246637 546785
Prep-5 1246545 546778 1246556 546778
Average 1246619 546512 1246684 546609.6
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Standard
deviation 842.6845 229.0557 152.608 200.1982
%RSD 0.067 0.041 0.012 0.036
8.6. Limit of detection and Quantification
8.6.1. Limit of detection for Irbesartan
The LOD is determined by the following formula
LOD=3.3 σ/S
=3.3(199.1213/10036)
=0.065µg/mL
8.6.2. Limit of Quantification for Irbesartan
The LOQ is determined by the following formula
LOQ=10 σ/S
=10(199.1213/10036)
=0.198 µg/mL
8.6.3. Limit of detection for Hydrochlorothiazide
The LOD is determined by the following formula
LOD=3.3 σ/S
=3.3(212.920/54427)
=0.013µg/mL
8.6.4. Limit of Quantification for Hydrochlorothiazide
The LOQ is determined by the following formula
LOQ=10 σ/S
=10(212.920/54427)
=0.039 µg/mL
The results were calculated and tabulated as follows
Table-9 Results of LOD and LOQ
Name of the Analyte LOD LOQ
Irbesartan 0.065µg/mL 0.198µg/mL
Hydrochlorothiazide 0.013µg/ml 0.039µg/mL
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8.7. Robustness
Robustness of the proposed method was done by making deliberate changes in the flow rate
and wavelength from the optimized conditions of the developed method and computing the
%RSD of the peak areas. The %RSD for Irbesartan and Hydrochlorothiazide at 0.8 ml/min
was found to be 0.87% and 0.67% respectively and at 1.2 ml/min they were found to be
0.67% and 0.23% respectively. At a wavelength of 255nm, the %RSDs for the drugs were
found to be 0.76%, 0.98% and at 259nm they were found to be 0.23% and 0.32%
respectively. As the %RSDs of peak areas and system suitability parameters were found to be
within the acceptance limit, the proposed method was said to be robust.
Table-10 Robustness data of Irbesartan and Hydrochlorothiazide
Parameter
Irbesartan Hydrochlorothiazide
Mean Rt
Mean Peak area
%RSD of peak areas
Mean Rt
Mean Peak area
%RSD of peak areas
Flow rate
(mL/min)
0.8 3.08 1286686 0.87 4.45 556789 0.67
1.0 2.86 1246767 0.71 3.94 546674 0.15
1.2 2.40 1278274 0.67 3.39 568798 0.23
Wavelength
(nm)
255 2.85 1244342 0.76 3.93 546323 0.98
257 2.86 1245127 0.65 3.94 546129 0.45
259 2.87 1247767 0.23 3.95 546189 0.32
As the peaks of analytes were well resolved and had no interference of excipients, it was
concluded that the proposed method was specific to the drugs under study. As all the
validation parameters studied, complied with the acceptance criteria, the proposed method
was said to be validated in accordance with ICH guidelines.
9. CONCLUSION
A simple and rapid reverse phase HPLC method was developed and validated according to
ICH guidelines for the simultaneous determination of Irbesartan and Hydrochlorothiazide in
tablet dosage form. The validation data signifies good specificity, accuracy, precision, and
reliability of the method. Because of its simplicity and low cost, the method can be used for
routine practices in the laboratories. The proposed method was found to be simple, precise,
accurate, rapid and specific for determination of Irbesartan and hydrochlorothiazide from
pure and its dosage forms. The mobile phase is simple to prepare and economical. The
sample recoveries in the formulation were in good agreement with their respective label
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claims and they suggested non-interference of formulation excipients in the estimation.
Hence, this method can be easily and conveniently adopted for routine analysis of Irbesartan
and hydrochlorothiazide in pure form and its dosage form and also can be used for
dissolution or similar studies.
10. REFERENCES
1) ICH Harmonized Tripartite guideline for validation of Analytical procedures Q2 (R1)
step 4 version Nov 2005.
2) David CL, Michael Webb. Pharmaceutical Analysis. London: Black well publishing;
1994. p. 2-4.
3) Gurdeep R. Chatwal, ShamK.Anand Instrumental methods of chemical analysis 5th
revised ed. Mumbai Himalaya publishing house 2009 p 2.570, 2.652.
4) B.K.Sharma. Instrumental methods of chemical analysis 25th Ed, Meerut, Goel
publishing house 2006 p 286-385.
5) Text book of Pharmaceutical Analysis, 7th edition by Asuthoshkar.
6) Hemamrutha, S.; Rambabu, R.; Vidhyadhara S. Simultaneous estimation and method
development of irbesartan and hydrochlorothiazide by RP HPLC .International Journal of
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7) Chabukswar, Aniruddha R.; Jagdale, Swati C.; Kuchekar, Bhanudas S.; Lokhande,
Pradeep D.; Shinde, Santosh N.; Ingale, Kunal D.; Kolsure, Anuja K. simultaneous
estimation of Hydrochlorothiazide and Irbesartan in tablet formulations by RP-HPLC-
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8) R. A. Mhaske .simultaneous determination of anti-hypertensive drugs Irbesartan,
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method. IJPSR 2012;3(4):1116-1123
9) Sane r. t. ; Francis m. ; Pawar s. ; et al. Determination of irbesartan in human plasma by
HPLC and a HPTLC method. Indian drugs coden indrba Source2003; 40(2): 104-110.
10) Tandogan, Ankara, Turkey; et al, assay for the antihypertensive drugs, irbesartan and
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11) Lara F. Tutunji, Maha F. Tutunji, Mamoun I. Alzoubi, Manal H. Khabbas, Adi I. Arida .;
developed and validated for the simultaneous determination of irbesartan and
hydrochlorothiazide in human plasma by liquid chromatography/tandem mass
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spectrometric method. Journal of pharmaceutical and biomedical analysis Molecules2012,
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12) Kishanta Kumar Pradhan .developed and validated for estimation of Irbesartan by
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13) V.P. Rane ,K.R. Patil , J.N. Sangshetti , R.D. Yeole and D.B. Shinde, developed and
validated for the quantitative simultaneous estimation of Irbesartan (IRB) and
hydrochlorothiazide (HCTZ) in combined pharmaceutical dosage form by stability-
indicating liquid chromatography method. J ChromatogrSci (2010); 48 (7): 595-600.
14) A. Ramprasad Reddy, GVS. Kumar*, SB. Puranik, PeerlaGiriprasad and KA. Sridhar
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2(4), 696-703.
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